Chia-Yi Kuan

Reduced Apoptosis and Cytochrome c–Mediated Caspase Activation in Mice Lacking Caspase 9

Caspases are essential components of the mammalian cell death machinery. Here we test the hypothesis that Caspase 9 (Casp9) is a critical upstream activator of caspases through gene targeting in mice. The majority of Casp9 knockout mice die perinatally with a markedly enlarged and malformed cerebrum caused by reduced apoptosis during brain development. Casp9 deletion prevents activation of Casp3 in embryonic brains in vivo, and Casp9-deficient thymocytes show resistance to a subset of apoptotic stimuli, including absence of Casp3-like cleavage and delayed DNA fragmentation. Moreover, the cytochrome c-mediated cleavage of Casp3 is absent in the cytosolic extracts of Casp9-deficient cells but is restored after addition of in vitro-translated Casp9. Together, these results indicate that Casp9 is a critical upstream activator of the caspase cascade in vivo.

Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene

Excitatory amino acids induce both acute membrane depolarization and latent cellular toxicity, which often leads to apoptosis in many neurological disorders,. Recent studies indicate that glutamate toxicity may involve the c-Jun amino-terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases. One member of the JNK family, Jnk3, may be required for stress-induced neuronal apoptosis, as it is selectively expressed in the nervous system,. Here we report that disruption of the gene encoding Jnk3 in mice caused the mice to be resistant to the excitotoxic glutamate-receptor agonist kainic acid: they showed a reduction in seizure activity and hippocampal neuron apoptosis was prevented. Although application of kainic acid imposed the same level of noxious stress, the phosphorylation of c-Jun and the transcriptional activity of the AP-1 transcription factor complex were markedly reduced in the mutant mice. These data indicate that the observed neuroprotection is due to the extinction of a Jnk3-mediated signalling pathway, which is animportant component in the pathogenesis of glutamate neurotoxicity.

The Jnk1 and Jnk2 protein kinases are required for regional specific apoptosis during early brain development.

The c-Jun NH2-terminal kinase (Jnk) family is implicated in apoptosis, but its function in brain development is unclear. Here, we address this issue using mutant mice lacking different members of the family (Jnk1, Jnk2, and Jnk3). Mice deficient in Jnk1, Jnk2, Jnk3, and Jnk1/Jnk3 or Jnk2/Jnk3 double mutants all survived normally. Compound mutants lacking Jnk1 and Jnk2 genes were embryonic lethal and had severe dysregulation of apoptosis in brain. Specifically, there was a reduction of cell death in the lateral edges of hindbrain prior to neural tube closure. In contrast, increased apoptosis and caspase activation were found in the mutant forebrain, leading to precocious degeneration. These results suggest that Jnk1 and Jnk2 regulate region-specific apoptosis during early brain development.

Mechanisms of programmed cell death in the developing brain

Programmed cell death (apoptosis) is an important mechanism that determines the size and shape of the vertebrate nervous system. Recent gene-targeting studies have indicated that homologs of the cell-death pathway in the nematode Caenorhabditis elegans have analogous functions in apoptosis in the developing mammalian brain. However, epistatic genetic analysis has revealed that the apoptosis of progenitor cells during early embryonic development and apoptosis of postmitotic neurons at later stage of brain development have distinct roles and mechanisms. These results provide new insight on the significance and mechanism of neural cell death in mammalian brain development.

Caspase‐3 is required for apoptosis‐associated DNA fragmentation but not for cell death in neurons deprived of potassium

Caspases are crucial effectors of the cell death pathway activated by virtually all apoptosis‐inducing stimuli within neurons and nonneuronal cells. Among the caspases, caspase‐3 (CPP32) appears to play a pivotal role and has been found to be necessary for developmentally regulated cell death in the brain. We have used mice lacking caspase‐3 (−/−CPP32) to examine its involvement in cultured cerebellar granule neurons induced to undergo apoptosis by potassium deprivation (K+). We find that, following K+ deprivation, neurons from −/−CPP32 mice die to the same extent as those from normal (+/+) mice. Although a small delay in the induction of cell death is observed in −/−CPP32 neurons, the rate of cell death is generally comparable to that of +/+ cultures. Though not critical for neuronal death, caspase‐3 is required for DNA fragmentation and chromatin condensation as judged by the absence of these apoptotic features in −/−CPP32 neurons. Boc.Asp.fmk, a pan caspase inhibitor, partially protects +/+ neurons from low‐K+‐mediated cell death and does so to the same extent in −/−CPP32 cultures, suggesting the involvement of a caspase other than caspase‐3 in cell death. However, the protective effect of boc.Asp.fmk is not seen beyond 24 hr, suggesting that the effect of caspase inhibition is one of delaying rather than preventing apoptosis. The more selective caspase inhibitors DEVD.fmk, IETD.fmk, and VEID.fmk fail to affect cell death, indicating that members inhibited by these agents (such as caspases ‐ 6 ,7, 8, 9 and 10) are also not involved in low‐K+‐mediated apoptosis. J. Neurosci. Res. 59:24–31, 2000

THE ROLES OF AUTOPHAGY IN CEREBRAL ISCHEMIA

Recent studies indicate the existence of autophagy in cerebral ischemia, but the functions of autophagy in this setting remain unclear. Here we discuss the role of autophagy in cerebral ischemia based on our own publication and the literature on this subject. We propose that oxidative and endoplasmic reticulum (ER) stresses in cerebral ischemia-hypoxia are potent stimuli of autophagy in neurons. We also reviewed evidence suggesting autophagosomes may have a shorter half-life in neurons and that a fraction of LC3 protein is degraded within autolysosomes, leading to a smaller detectable amount of LC3-II in the brain while there are clear indications of on-going autophagy. Finally, we suggest autophagy is an important modifier of cell death and survival, interacting with necrosis and apoptosis in determining the outcomes and final morphology of deceased neurons.

JNK3 contributes to c-Jun activation and apoptosis but not oxidative stress in nerve growth factor-deprived sympathetic neurons.

The stress activated protein kinase pathway culminates in c‐Jun phosphorylation mediated by the Jun Kinases (JNKs). The role of the JNK pathway in sympathetic neuronal death is unclear in that apoptosis is not inhibited by a dominant negative protein of one JNK kinase, SEK1, but is inhibited by CEP‐1347, a compound known to inhibit this overall pathway but not JNKs per se. To evaluate directly the apoptotic role of the JNK isoform that is selectively expressed in neurons, JNK3, we isolated sympathetic neurons from JNK3‐deficient mice and quantified nerve growth factor (NGF) deprivation‐induced neuronal death, oxidative stress, c‐Jun phosphorylation, and c‐jun induction. Here, we report that oxidative stress in neurons from JNK3‐deficient mice is normal after NGF deprivation. In contrast, NGF‐deprivation‐induced increases in the levels of phosphorylated c‐Jun, c‐jun, and apoptosis are each inhibited in JNK3‐deficient mice. Overall, these results indicate that JNK3 plays a critical role in activation of c‐Jun and apoptosis in a classic model of cell‐autonomous programmed neuron death.

Jun NH2-terminal kinase (JNK) prevents nuclear β-catenin accumulation and regulates axis formation in Xenopus embryos

Jun NH2-terminal kinases (JNKs) regulate convergent extension movements in Xenopus embryos through the noncanonical Wnt/planar cell polarity pathway. In addition, there is a high level of maternal JNK activity spanning from oocyte maturation until the onset of gastrulation that has no defined functions. Here, we show that maternal JNK activation requires Dishevelled and JNK is enriched in the nucleus of Xenopus embryos. Although JNK activity is not required for the glycogen synthase kinase-3-mediated degradation of β-catenin, inhibition of the maternal JNK signaling by morpholino-antisense oligos causes hyperdorsalization of Xenopus embryos and ectopic expression of the Wnt/β-catenin target genes. These effects are associated with an increased level of nuclear and nonmembrane-bound β-catenin. Moreover, ventral injection of the constitutive-active Jnk mRNA blocks β-catenin-induced axis duplication, and dorsal injection of active Jnk mRNA into Xenopus embryos decreases the dorsal marker gene expression. In mammalian cells, activation of JNK signaling reduces Wnt3A-induced and β-catenin-mediated gene expression. Furthermore, activation of JNK signaling rapidly induces the nuclear export of β-catenin. Taken together, these results suggest that JNK antagonizes the canonical Wnt pathway by regulating the nucleocytoplasmic transport of β-catenin rather than its cytoplasmic stability. Thus, the high level of sustained maternal JNK activity in early Xenopus embryos may provide a timing mechanism for controlling the dorsal axis formation.

Programmed cell death in mouse brain development.

Among the basic cellular events that shape the developing brain, programmed cell death (also called apoptosis) plays an essential role (Cowan et al. 1984; Oppenheim 1991). Although cell death during the development of the vertebrate nervous system was first described by anatomists of the nineteenth century, a mechanistic understanding of these events started only in the latter half of this century through two lines of research. First, the identification of nerve growth factor (NGF) by R. Levi-Montalcini and colleagues showed that cells died if deprived of trophic molecules and thus established the foundation for the “trophic theory” of neural development. Second, H. R. Horvitz pioneered genetic studies of programmed cell death in the nematode Caenorhabditis elegans and elucidated a genetic pathway involved in this process. Because the targeted disruption of specific genes in the mouse can now be performed, we can test directly whether the same cell death machinery is conserved in mammals. Moreover, the similarity of the organization and development of the central nervous system between the mouse and higher primates makes it an ideal experimental system for understanding the roles of programmed cell death in normal human brain development and congenital malformations. The present chapter focuses on the recent gene targeting studies elucidating the evolutionarily conserved cell death machinery in the context of mouse brain development.

Cell Death in Mammalian Development

Physiological mechanisms of cell death are required throughout the development and adulthood of all multicellular organisms (1,2). From primitive multicellular organisms to higher vertebrates, well-orchestrated cell-death events are critical for the removal of superfluous cells as a vital part of tissue sculpting during development. Physiological cell death enables the elimination of unwanted extra cells to maintain cellular homeostasis in developed adults, as well as the elimination of organisms of harmful cells or cells with serious cellular or genomic damage. Apoptosis—physiological cell death—is characterized by a distinct set of morphological and biochemical features including chromatin condensation, internucleosomal DNA fragmentation, and perhaps most important, cell-surface alterations, which signal for the rapid recognition and engulfment of apoptotic cells by neighboring phagocytic cells, thus avoiding the induction of any pathological reactions (3).